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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1+ cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1+ fetal liver cells. Therefore, we isolated Thy-1+ and Thy-1 cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1+. The vast majority of alpha fetoprotein+, albumin+, cytokine-19+, and E-cadherin+ cells were found in cultured Thy-1 cells, whereas nearly all CD45+ cells were in the Thy-1+ fraction. In normal rat liver, transplanted Thy-1+ cells produced only rare, small DPPIV+ cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1+ fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1 fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell–derived factor (SDF)–1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1+ and Thy-1 fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1α expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy–treated liver, and this correlated with liver repopulation by Thy-1+ cells. Conclusion: Highly enriched Thy-1+ ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1 stem/progenitor cells, which repopulate the normal adult liver. (HEPATOLOGY 2007.)

Liver transplantation is currently the accepted therapy for patients with end-stage liver disease. Because there is a serious shortage in the supply of donor organs,1 recent studies have focused on identifying cells that can proliferate after transplantation into diseased recipient liver and restore liver mass.2 Several animal models have been established in which transplanted hepatocytes effectively replace the recipient liver, but this requires both extensive and continuous liver injury and significant selection pressure favoring the proliferation/survival of transplanted cells.3–9 However, opportunities to transplant hepatocytes under these circumstances will be encountered infrequently in clinical medicine. Therefore, there is substantial interest in identifying candidate stem or progenitor cells for liver cell transplantation.10

One possible source of stem or progenitor cells is the oval cell, which is induced to proliferate in rats after treatment with 2-acetaminofluorine (2-AAF)/partial hepatectomy (PH)11, 12 or d-galactosamine.13, 14 Oval cells are the progeny of canal of Hering cells and differentiate into hepatocytes.15, 16 They express a variety of stem cell markers: c-kit,17 CD34,18 fms-like tyrosine kinase receptor,19 and leukemia inhibitory factor.20 Petersen and coworkers21 showed that rat oval cells express Thy-1, a well-known hematopoietic stem cell marker,22 and they also reported that bone marrow (BM) cells differentiated into oval cells and hepatocyte-like cells after transplantation into the liver.23 Subsequently, Avital et al.24 isolated β2m/Thy-1+ bone marrow cells (bone marrow–derived hepatocyte stem cells, or BDHSCs) that express liver-specific genes and functions in cell culture. After transplantation into rat livers under strong selection pressure and continuous host liver injury, BDHSCs integrated into the host parenchyma and differentiated into hepatocytes with significant liver repopulation.25

Subsequent studies focused on mouse oval cells induced in the liver by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Petersen et al.26 reported that DDC induces at least 4 distinct fractions of nonparenchymal cells, one of which expresses hematopoietic stem cell markers Sca-1 and CD34, the mature hematopoietic lineage marker CD45, and the liver progenitor cell marker α-fetoprotein (AFP). However, Thy-1 expression was not mentioned in these studies.26 Wang et al.27 subsequently showed that DDC-induced mouse oval cells are not derived from hematopoietic stem cells, although they do effectively repopulate the fumarylacetoacetate hydrolase–null mouse liver.

On embryonic day (ED) 14, rat fetal liver also contains a small subpopulation of hepatic specified bipotent cells that, after transplantation, engraft, proliferate, differentiate into hepatocytes and bile duct cells, and stably repopulate normal adult liver.28–30 There is evidence that Thy-1+ oval cells are in developing fetal liver,31, 32 and therefore, it was of much interest to further characterize these cells and determine whether they represent a potential source for cell-based therapies. In the present study, we isolated Thy-1+ and Thy-1 cell populations from rat ED14 fetal liver, characterized these cells in vitro, and compared their ability to repopulate a normal host liver. In contrast to Thy-1 fetal liver cells, Thy-1+ fetal liver cells are unable to repopulate the normal host liver, but they can repopulate retrorsine-treated rat liver, an animal model in which host hepatocyte proliferation is markedly impaired.5 These findings suggest that Thy-1+ fetal liver cells at ED14 represent a population of progenitor cells that can repopulate only a massively injured liver, whereas Thy-1 fetal liver cells are stem/progenitor cells that exhibit greater proliferation potential and can repopulate the normal adult liver.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Animals and Animal Treatment

Pregnant, ED14 dipeptidyl peptidase IV (DPPIV)+ F344 rats were purchased from Taconic Farms, Germantown, NY. DPPIV F344 rats were provided by the Liver Research Center, Albert Einstein College of Medicine. For some studies, DPPIV F344 rats weighing 90–140 g were treated with retrorsine, as described.5 Animal studies were conducted under protocols approved by the Animal Care Use Committee of Albert Einstein College of Medicine.

Fetal Liver Cell Isolation

Unfractionated fetal liver stem/progenitor cells were isolated from ED14 fetal livers of DPPIV+ pregnant F344 rats, as described previously.29 For large-scale preparation of ED14 fetal liver cells, we used 5 pregnant rats containing 49 embryos from which we obtained 7.6 × 106 Thy-1+ cells and 84 × 106 Thy-1 cells.

Immunoselection of Thy-1+ Fetal Liver Cells

After isolation, cells were incubated with mouse anti-Thy-1 (BD Biosciences, San Jose, CA) at 4°C for 20 minutes. Cells were washed twice in Hanks' balanced salt solution (HBSS) containing 0.8 mM MgCl2, 20 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated with rat antimouse IgG1 bound to magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA). After washing, the cell suspension was passed through an LS column placed in a MidiMACS Separator (Miltenyi). Thy-1+ cells were retained in the magnetic field (column); Thy-1 cells passed through the column. Cell fractions were washed twice in HBSS. Cell viability was >94% and >92% in the Thy-1+ and Thy-1 fractions, respectively.

Cell Culture

After isolation and purification, 0.35 × 106 to 1.0 × 106 Thy-1+ and Thy-1 fetal liver cells were plated on gelatin-coated 2-well chamber slides. Plated cells were incubated in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin for 24 hours, after which the medium was switched to hormonally defined hepatocyte growth medium plus 10 ng/mL epidermal growth factor (EGF) and 10 ng/mL fibroblast growth factor (FGF).33, 34 On days 2 and 5 after cell plating, chamber slides were used for immunohistochemical analysis or cultured cells were released from the chamber slides, using trypsin/EDTA, and fixed to cytospin slides.

Antibodies Used for Immunohistochemical Analysis

Primary Antibodies.

Mouse anti-Thy-1 (BD), rabbit anti-AFP, and rabbit anti-Ki-67 came from LabVision Corp. (Fremont, CA); mouse anti-CK-19 was supplied by Progene Biotechnik GmbH (Heidelberg, Germany); rabbit anti-Alb came from ICN Biochemicals Inc. (Aurora, OH); mouse anti-E-cadherin came from BD; rabbit anti-CXCR4 was supplied by Torrey Pines Biolabs, Inc. (Houston, TX); mouse anti-CD45 came from AbD Serotec (Raleigh, NC); sheep anti-AFP was supplied by Nordic Immunological Laboratories (Tilburg, NL); and mouse anti-SDF-1α came from R&D Systems, Inc. (Minneapolis, MN).

Secondary Antibodies:

Cy3-conjugated donkey antimouse IgG, Cy3-conjugated donkey antirabbit IgG, Cy2-conjugated donkey antirabbit IgG, Cy2-conjugated donkey antisheep IgG, and Cy5-conjugated donkey antisheep IgG came from Jackson Immunoresearch Laboratories (West Grove, PA); biotin-conjugated goat antimouse IgG2 came from Nordic; and streptavidin-Cy2 and TRITC-conjugated rabbit antimouse IgG1 came from Rockland Inc. (Gilbertsville, PA).

Fetal Liver Cell Transplantation

After magnetic microbead cell sorting, Thy-1+ cell fractions (2.0 × 106 to 2.4 × 106 cells) were transplanted into normal rats in conjunction with two-thirds PH (n = 8). For direct comparison, equal numbers (2.0 × 106 to 2.4 × 106 cells) of Thy-1 fetal liver cells (n = 4) were used as donor cells.

In additional experiments, after PH, 0.75 × 106 to 1.2 × 106 Thy-1+ or 10 × 106 Thy-1 fetal liver cells were infused through the portal vein into the liver of retrorsine-treated rats (n = 8).

At various times after cell transplantation, rats were sacrificed and liver tissue samples snap-frozen in 2-methylbutane at −70°C.

DPPIV Detection

DPPIV expression was determined by enzyme histochemistry, and the percentage of liver repopulation quantified, as previously reported.29 Additional information is provided in Supplementary Experimental Procedures and Supplementary Table 1.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Enrichment and Characterization of Fetal Liver Cells.

Fetal liver cells were selected for Thy-1 expression using immunomagnetic bead sorting (magnetic cell sorting, or MACS). The purity of Thy-1+ selected fetal liver cells was determined by immunohistochemistry. Unfractionated fetal liver cells contained 5.2% ±1.5% Thy-1+ cells (Fig. 1A). After MACS enrichment for Thy-1, 87.2% ± 1.9% of the cells were Thy-1+ (Fig. 1B). No Thy-1+ cells were found in the immunodepleted Thy-1 fraction (results confirmed by RT-PCR, Fig. 1C).

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Figure 1. Enrichment of rat ED14 fetal liver cells. To purify fetal liver cells, magnetic bead sorting (MACS) was used to select Thy-1+ cells. Thy-1+ cells made up 5.2% of unfractionated fetal liver cells (A); after enrichment with magnetic microbeads, this increased to 87.2% (B). Cells were immunostained for Thy-1 (red fluorescence). DAPI was used to stain nuclei (blue). Data are shown as merged images. (Original magnification ×400.) (C) RT-PCR analysis showed that none of the Thy-1+ cells were in the Thy-1 fraction after enrichment with anti-Thy-1 magnetic microbeads.

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To evaluate the stem/progenitor status of microbead-sorted cells, Thy-1+ and Thy-1 fractions were analyzed by immunohistochemistry for AFP, albumin, cytokeratin (CK)−19, and E-cadherin. In the Thy-1+ fraction, cells were positive only rarely for AFP (0.1% ± 0.1%), albumin (0.2% ± 0.2%), CK-19 (0.8% ± 0.3%), or E-cadherin (0.1% ± 0.1%); see Fig. 2A and Supplementary Fig. 1. These results were confirmed by Western blots showing much lower AFP and CK-19 expression in Thy-1+ liver cell fractions than in Thy-1 fetal liver cell fractions (Fig. 2C). In contrast, in the Thy-1 fraction, 4.3% ± 0.7% of the cells were AFP+, 4.0% ± 0.5% were albumin (Alb)+, 2.5% ± 0.5% were CK-19+, and 4.9% ± 2.2% were E-cadherin+ (Fig. 2A and Supplementary Fig. 1). RT-PCR analysis of a representative group of liver-specific genes, stem/progenitor cell markers, and epithelial markers showed that the Thy-1 cell fraction best exhibited the gene expression profile expected for liver stem/progenitor cells (Fig. 2D).

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Figure 2. Phenotypic characterization of the Thy-1+ and Thy-1 fractions after cell sorting. ED14 fetal liver cells were isolated and enriched using anti-Thy-1 magnetic microbeads. (A) Thy-1+ and Thy-1 liver cell fractions were characterized by immunohistochemical analysis for expression of AFP, albumin, CK-19, and E-cadherin, and the percentages were calculated. (B, upper panel) AFP staining (red fluorescence); an example of a Thy-1 cell fraction. (B, lower panel) CK-19 detection (green); an example of a Thy-1 cell fraction. DAPI was used for nuclear staining (blue). Data are shown as merged images. (Original magnification ×900.) Data were confirmed by Western blot analysis (C). (D) Gene expression characteristics of Thy-1 and Thy-1+ liver cell fractions, as determined by RT-PCR with total RNA isolated from these cell fractions.

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Further characterization of Thy-1+ cells was performed by dual immunohistochemistry for AFP/Thy-1 and CD45/Thy-1. Of the 0.1% AFP+ cells in the Thy-1+ fraction, 78.4% ± 9.7% were positive for both AFP and Thy-1 (Fig. 3A-D). Therefore, the maximum potential contamination of the Thy-1+ cell fraction by Thy-1/AFP+ cells was 0.02%. Because Thy-1 is also expressed on hematopoietic cells,22 we determined whether Thy-1+-selected fetal liver cells also expressed CD45. Although the intensity of CD45 expression in Thy-1+ fetal liver cells was very low, approximately 30% of Thy-1+ cells were positive for CD45 (Fig. 3E-H).

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Figure 3. Dual immunohistochemical analysis of sorted fetal liver cell isolates. After isolation of ED14 fetal liver cells, cells were enriched using anti-Thy-1 magnetic microbeads. Separated cells (Thy-1+ fraction) were characterized using antibodies for AFP/Thy-1 (A, B) and CD45/Thy-1 (E, F). DAPI was used to stain nuclei (C, G). (Original magnification ×900.)

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Properties of Thy-1+ Fetal Liver Cells in Cell Culture.

Figure 4 shows the morphological characteristics of Thy-1+ and Thy-1 cells in culture. On day 2 after plating, both cell fractions showed 2 cell types, epithelial and nonepithelial cells. In Thy-1+ cell cultures, most attached cells were scattered and exhibited a fibroblastoid morphology (referred to as nonepithelial; Fig. 4A). A minority of cells formed small epithelial clusters expressing E-cadherin (1.3% ± 0.6%; Fig. 4A,C). In contrast, most Thy-1 cells in culture were epithelial (51% ± 3.6% E-cadherin+) and formed clusters (Fig. 4B,D). Both Thy-1+ and Thy-1 cells expressed nuclear Ki-67 (45.1% ± 1.8% and 48.4% ± 3.4%, respectively), indicating active proliferation (Fig. 4E-H), including AFP+/Ki67+ cells (Fig. 4I,J). On day 5, the epithelial cell clusters increased progressively in size and cell number in both the Thy-1+ and the Thy-1 cell cultures (Fig. 4K-P); however, cluster size of Thy-1 cells was significantly larger than that of Thy-1+ cells (Fig. 4L,N vs. K,M).

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Figure 4. Comparison of Thy-1+ and Thy-1 fetal liver cells in culture. After enrichment with anti-Thy-1 magnetic microbeads, 0.35 × 106 to 1.0 × 106 Thy-1+ or Thy-1 cells were plated on gelatin-coated 2-well chamber slides and cultured for up to 5 days. (A-J) Equal numbers of Thy-1+ and Thy-1 fetal liver cells were cultured for 2 days. (A, B) Small epithelial cell colonies expressing E-cadherin (green fluorescence) are highlighted by arrows. (C, D) E-cadherin positive cell clusters are shown at higher magnification. Nuclei were identified by DAPI (blue). (E, F) Cultured Thy-1 and Thy-1+ cells are strongly positive for Ki-67 (red). (G, H) Cells were released from chamber slides and fixed to cytospins. Cytospins were stained for Thy-1 (red) and Ki-67 (green). Nuclei were identified by DAPI (blue). (I, J) AFP+ epithelial cell clusters (green) show proliferative activity in both cell fractions (Ki-67; red). (K-P) On day 5 after cell plating, cells were fixed on chamber slides and stained for Thy-1/AFP, Thy-1/albumin, or Thy-1/CD45 (red/green). Cells in the Thy-1+ fraction formed smaller AFP+ (K) and albumin+ cell colonies (M) compared with those in the Thy-1 fetal liver cell fraction (L, N). Most CD45+ cells were found in cell cultures of Thy-1+ fetal liver cells (O, see arrows). However, none of the epithelial cell colonies were positive for CD45 in both cultured fractions (O, P); insets show CD45+ cells in each fraction at higher magnification. DAPI was used for staining of nuclei (blue). Data are shown as merged images. (Original magnification ×200 [A, B, E, F], ×400 [C, D, G-P], ×900 [O, P: insets].)

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On day 5, Thy-1+ and Thy-1 cells were fixed and stained on chamber slides. In Thy-1+ cell cultures, few epithelial cell clusters were positive for AFP (Fig. 4K). In addition, all epithelial colonies expressed albumin (Fig. 4M). Thy-1 expression decreased in Thy-1+ cell cultures on day 5 (Fig. 4K,M,O). However, double immunohistochemistry for AFP/Thy-1 and albumin/Thy-1 still showed AFP+ and albumin+ clusters coexpressing Thy-1 (Fig. 4K,M). These results confirm that there is indeed a subpopulation of Thy-1+ cells that expresses hepatic epithelial cell markers, and these cells can be expanded in cell culture.

To further characterize cultured Thy-1+ cells in vitro, we analyzed expression of hematopoietic marker CD45 by double immunohistochemistry with Thy-1. None of the epithelial cell colonies were positive for CD45. However, a substantial number of nonepithelial cells expressed CD45 and were therefore of hematopoietic origin (Fig. 4O). In Thy-1 cell cultures, most epithelial colonies expressed AFP (Fig. 4L) and albumin (Fig. 4N). Cells in the Thy-1 fraction rarely were CD45+, and these cells exhibited a nonepithelial morphology (Fig. 4P). In the Thy-1 fraction, Thy-1 expression was not induced on day 5 after cell plating (Fig. 4L,N,P).

One possible explanation for these results is that cell debris attached to the surface of growing cells could produce false-positive results during double immunohistochemistry. Therefore, to confirm our observations, Thy-1+ cells cultured for 2 days were released from chamber slides using trypsin/EDTA, washed twice, and fixed to cytospin slides. Some Thy-1+ cells still expressed albumin (Fig. 5A,D) or CD45 (Fig. 5B,E), whereas other Thy-1+ cells were negative for both markers (Fig. 5A,B,D,E). None of the cultured Thy-1+ fetal liver cells coexpressed albumin and CD45 (Fig. 5C,F).

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Figure 5. Phenotypic characterization of Thy-1+ cells in vitro. After enrichment by magnetic microbeads, Thy-1+ cells were cultured, and on day 2 after cell plating, cells were released from the chamber slides, using trypsin/EDTA digestion, and wer fixed to cytospin slides. Thy-1+ cells (red fluorescence; A, B, D, E) expressed albumin (green; A, D) and CD45 (green; B, E) or were negative for both markers. Double immunohistochemistry for albumin (red; C, F) and CD45 (green; C, F) showed that none of the cultured cells coexpressed albumin and CD45. Nuclei were identified by DAPI (blue). Data are shown as merged images. (Original magnification ×600 [A-C] and ×900 [D–F].)

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Repopulation of Rat Liver by Anti-Thy-1 Magnetic Bead–Selected ED14 Fetal Liver Cells.

The proliferative activity and differentiation potential of DPPIV+ ED14 fetal liver Thy-1+ cells was investigated further by cell transplantation into DPPIV mutant rats. In normal adult liver, transplanted Thy-1+ fetal liver cells produced only rare DPPIV+ hepatocytic clusters and did not repopulate the liver (Fig. 6A). In contrast, an equal number of Thy-1 fetal liver cells substantially repopulated the normal liver, with differentiation into both hepatocytes and bile duct epithelial cells (Fig. 6B).

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Figure 6. Repopulation of normal or retrorsine-treated rat liver with anti-Thy-1 magnetic microbead–selected ED14 fetal liver cells. Fetal liver cells from DPPIV+ F344 rats were isolated and enriched using magnetic cell sorting, followed by cell transplantation into DPPIV rats in conjunction with two-thirds PH. After sacrifice of the animals, transplanted cells were detected by DPPIV enzyme histochemistry. (A) In the absence of retrorsine treatment, transplanted Thy-1+ fetal liver cells (2.0 × 106 to 2.4 × 106 cells) produced only a small number of single DPPIV+ cells or cell clusters, and very few of these clusters exhibited a hepatocytic phenotype. (B) In contrast, after transplantation of an equal number of Thy-1 fetal liver cells into normal liver, substantial repopulation occurred, and transplanted cells differentiated into both hepatocytes and bile duct cells (highlighted by arrows). Two examples of transplanted Thy-1 cells that differentiated into bile duct epithelial cells are shown as insets at higher magnification (B). (C) After transplantation of Thy-1+ cells (0.75 × 106 to 1.2 × 106 cells) into liver of retrorsine-treated rats, a small number of Thy-1+ cells proliferated extensively and differentiated into hepatocytes. (D) In contrast, essentially total liver replacement was achieved by transplanted Thy-1 fetal liver cells. Animals were sacrificed 4 months after cell transplantation (A-D). (Original magnification ×100, insets × 600 [A, B].)

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Because Thy-1+ fetal liver cells did not repopulate normal adult liver, they were transplanted into retrorsine/PH-treated rats, which induces liver repopulation by transplanted hepatic cells, regardless of whether they are progenitor cells or adult hepatocytes.5, 35 For these experiments, 0.75 × 106 to 1.2 × 106 Thy-1+ cells (17-fold enriched by MACS) versus 10 × 106 Thy-1 cells (not enriched) were transplanted. If the repopulation capacities of these 2 cell fractions were equivalent, then the effective number of putative liver-repopulating cells transplanted from the Thy-1+ fraction would be twice that used from the Thy-1 fraction. However, in retrorsine/PH-treated rats, Thy-1+ cells produced only a small number of very large clusters composed primarily of hepatocytes (Fig. 6C). In contrast, Thy-1 cells repopulated the entire liver (Fig. 6D). As shown in Table 1, repopulation by transplanted Thy-1+ fetal liver cells in 1 month ranged up to 9.0% and at 4 months up to 23.5% (Table 1).

Table 1. Repopulation of Liver by Thy-1+ Fetal Liver Cells in Retrorsine-Treated Rats
Number of ratsTime after cell transplantationRepopulation (%)*Phenotype of cell clusters
  • *

    Two sections from different areas/lobes of each rat liver were scanned, and repopulation was determined by computerized procedure (see Materials and Methods section).

  • The vast majority of DPPIV+ clusters exhibited an hepatocytic phenotype.

43-4 weeks0.2%–9.0%Hepatocytic
44 months4.6%–23.5%Hepatocytic

Role of CXCR4 and SDF-1α in Liver Repopulation by Thy-1+ and Thy-1 Fetal Liver Cells.

To evaluate the mechanism by which Thy-1+ and Thy-1 fetal liver epithelial cells repopulate the adult rat liver, we studied CXCR4 expression in cells to be transplanted and stromal cell–derived factor (SDF)–1α expression in recipient host liver. As shown in Fig. 7, both the Thy-1+ and Thy-1 cells expressed CXCR4 (Fig. 7A,B,G,H). By triple immunohistochemistry, some Thy-1+ cells expressed both CXCR4 and AFP (Fig. 7C–F), indicating that they were fetal liver epithelial progenitor cells. Similarly, CXCR4+/AFP+ epithelial cells were identified in the Thy-1 cell fraction (Fig. 7I-L). As shown in Fig. 8, there was very low SDF-1α expression in the bile ducts of normal adult rat liver (Fig. 8A), which increased after PH (Fig. 8B). SDF-1α expression was slightly higher in retrorsine-treated rat liver than in normal rat liver (Fig. 8C) and increased dramatically after PH, in which case SDF-1α-positive oval cells were also identified (Fig. 8D). These findings suggest that increased SDF-1α expression by bile duct epithelial cells and oval cells after retrorsine/PH treatment mediates repopulation of the liver by Thy-1+ fetal liver epithelial cells.

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Figure 7. CXCR4 expression in Thy-1+ and Thy-1 fetal liver cells. CXCR4 was highly expressed in both Thy-1+ and Thy-1 cell fractions (A,B and G,H). AFP expressing Thy-1+ (C-F) and Thy-1 (I-L) fetal liver epithelial cells were positive for CXCR4. (Original magnification ×200 [A,G], ×900 [B-F, H-L].)

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Figure 8. SDF-1α expression in normal and retrorsine-treated rat liver before and after partial hepatectomy (PH). Immunohistochemistry for SDF-1α was performed in normal (A) and retrorsine-treated (C) rat liver and on day 3 after two-thirds PH in normal (B) and retrorsine-treated (D) rat liver. Areas of oval cell proliferation are highlighted by arrows. Hepatectomized normal and retrorsine-treated liver tissues were used as controls (A, B). (Original magnification ×100.)

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Putative hepatic stem/progenitor cells have been identified in adult rodent and human liver by coexpression of stem cell markers (e.g., Thy-1, CD34, and c-kit) and hepatic lineage markers (AFP, Alb, CK-18, and CK-19).21, 26, 36, 37 Thy-1+ cells isolated from rat fetal liver also express the products of the liver-specific genes CK-18, AFP, and Alb.31, 38 In addition, Lazaro et al.39 reported that after plating human fetal hepatocytes (AFP+/Alb+/CK-19+), blast-like cell clusters appeared and showed expression of oval cell markers (Thy-1, CD34, and OV-6), hepatoblast markers (78.75, 78.26), Alb, and CK-19. Because Thy-1+ human fetal liver cells coexpress hepatocellular and biliary proteins and mRNA (AFP, human nuclear factor–4α, CK-18, CK-19), Masson et al.32 suggested Thy-1 as a marker of hepatic stem cells in fetal liver.

In the present study, we isolated Thy-1+ and Thy-1 cell populations from rat ED14 fetal liver by immunomagnetic bead selection for Thy-1. Using immunohistochemistry, Western blots and RT-PCR, we identified a small number of AFP+, Alb+, CK-19+, and E-cadherin+ cells in the Thy-1+ cell fraction. Our results agree with those reported by Fiegel et al.,31 who showed that Thy-1+ rat fetal liver cells that coexpress hepatic lineage markers (CK-18) represent a small fraction of total fetal liver cells (0.5%-1.0%). This is in contrast to the results of a study by Hoppo et al.,40 who reported that Thy-1+ mouse fetal liver cells are negative for AFP, CK-19, and Alb. Consistent with the findings in other studies,13, 38, 40 we found that most cells expressing hepatic markers (AFP+, CK-19+, Alb+) were in the Thy-1 fetal liver cell fraction.

To investigate the growth and differentiation potential of Thy-1+ fetal liver cells compared to Thy-1 cells, we cultured both fractions in hormonally defined hepatocyte growth medium containing EGF and FGF for up to 5 days. The growth potential of Thy-1+ fetal liver cells was lower than that of Thy-1 cells. This contrasts with the results of the study by Fiegel et al.,41 who reported a significantly higher growth potential of Thy-1+ rat fetal liver cells than of Thy-1 fetal liver cells.

Double immunohistochemistry for Thy-1/AFP showed that most (∼80%) AFP+ cells in the Thy-1+ fraction coexpressed Thy-1 and therefore did not represent contaminants from the Thy-1 cell fraction. In addition, immunohistochemistry for Thy-1/CD45 and Alb/CD45 showed that a substantial number of Thy-1+ cells expressed the hematopoietic surface marker CD45; however, none of these cells coexpressed Alb. Therefore, Thy-1+ fetal liver cells expressing hepatic lineage markers represent a separate and unique cell population, distinct from hematopoietic stem cells.

Because transplantation into the liver is a definitive method for identifying candidate stem or progenitor cells based on their functional capacity,10, 42 we transplanted both Thy-1+ and Thy-1 cell fractions to determine their hepatic stem/progenitor cell potential. Because rat ED14 fetal liver contains both lineage-committed (unipotential) progenitor cells and lineage-uncommitted (bipotential) stem/progenitor cells,28 we used 2 models to assess repopulation potential of Thy-1+ and Thy-1 fetal liver cells: normal adult rat liver and retrorsine/PH-treated rat liver. In normal liver, only stem or stem/progenitor cells effectively repopulate the liver.29, 30 However, in retrorsine/PH-treated rats, all cells with hepatocytic potential (mature hepatocytes, unipotent hepatocyte progenitor cells, and bipotent stem/progenitor cells) can repopulate the liver.29

In the present study, the Thy-1+ cell fraction did not repopulate the normal liver but did repopulate the retrorsine/PH-treated liver, whereas Thy-1 cells repopulated both models. Approximately 80% of the AFP+ cells in the Thy-1+ fraction were positive for both Thy-1 and AFP. We believe these cells are less effective at repopulating retrorsine/PH-treated liver than are Thy-1 fetal liver stem/progenitor cells. However, we cannot rule out the possibility that very low levels of Thy-1 cells contained in the Thy-1+ fraction (∼0.02% of total cells) contributed to liver repopulation by this cell population.

As studied extensively with hematopoietic stem cells, the CXCR4/SDF-1 axis is involved in homing, engraftment, and repopulation of the BM by transplanted cells.43 This same mechanism has been reported for hematopoietic stem and progenitor cell engraftment in the liver.44 Petersen and coworkers45 reported CXCR4 expression in proliferating oval cells and SDF-1α in hepatocytes in the 2-AAF/carbon tetrachloride (CCl4) rat model of liver injury/regeneration. Mavier et al.46 reported expression of both CXCR4 and SDF-1α in activated oval cells in the same model, and most recently Zweibel et al.47 reported CXCR4 expression by Thy-1+ ED14 fetal liver cells. In addition, Kollet et al.44 demonstrated SDF-1α expression in bile duct epithelial cells and increased expression of SDF-1α in rodent liver after acute injury with CCl4. In the present study, we showed strong expression of CXCR4 in both Thy-1+ and Thy-1 rat fetal liver progenitor cells, so this does not explain the difference in behavior of these cells after transplantation. However, SDF-1α expression was increased substantially in bile ducts and oval cells after retrorsine/PH treatment, and this correlated with the ability of Thy-1+ fetal liver progenitor cells to repopulate retrorsine-treated rat liver. These results are consistent with a recent study by Zheng et al., which showed that increased expression of SDF-1α in 2-AAF/PH-treated rats leads to increased proliferation of oval (progenitor) cells.48

Various studies have reported fusion of BM stem cells to hepatocytes in liver repopulation models in which there is extensive liver injury and strong selection for survival of transplanted cells,49, 50 including a study in a mouse retrorsine model in which both fusion and nonfusion events were reported.51 Fusion appears to be a specific property of myeloid cells and myeloid progenitor cells52, 53 but has not been reported with fetal liver progenitor cells or adult hepatocytes. In other studies with BM-derived hematopoietic stem cells or mesenchymal stem cells, generally low levels of plasticity or transdifferentiation into an hepatocytic phenotype have been reported.54–58 However, in the DPPIV model, we are not able to study fusion because it is not possible to separate fetuses according to their sex prior to cell isolation and transplantation.

In conclusion, Thy-1+ ED14 fetal liver cells represent a separate population of hepatic progenitor cells in ED14 fetal rat liver that are able to proliferate after cell transplantation and differentiate into hepatocytes. However, this requires extensive liver injury and substantial selective pressure, which are not required for repopulation by Thy-1 ED14 stem/progenitor cells. From the results of the present study, it is clear that the bulk of the liver repopulation capacity of fetal liver epithelial cells resides in the Thy-1 cell population.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

The authors thank Anna Caponigro and Emily Bobe for secretarial assistance.

Note Added in Proof:

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

A recent study by Corcelle et al (Exp. Cell Res. 312:2826-2836, 2006) reported two distinct populations of oval cells in the liver of 2-AAF/CCI4 treated rats, one derived from the biliary epithelium (OV6+/CK-19+/Rab 3b+ cells) and the other from the bone marrow (c-kit+ cells). After cell culture, the latter expressed Thy-1 and low levels of alb mRNA and may be related to our Thy-1+ cells derived from fetal liver.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Note Added in Proof:
  8. References
  9. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY Web site ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

FilenameFormatSizeDescription
jws-hep.21775.tbl1.doc35K Supplementary Table 1:Primer Sequences used for RT-PCR
jws-hep.21775.fig1.tif6208K Supplementary Fig. 1:Albumin and E-cadherin expression in the Thy-1 + and Thy-1- fractions after selection with anti-Thy-1 magnetic microbeads. Immunohistochemical analysis for albumin showed lower albumin expression (red) in Thy-1 + cell fractions ( A & C ) compared to the Thy-1- fetal liver cell fraction ( B & D ). Immunohistochemical analysis for E-cadherin expression (green) showed similar results, as only rare E-cadherin positive cells were detected within the Thy-1 + fetal liver cell fraction ( E ), whereas many E-cadherin positive cells were found in the Thy-1- fraction ( F ). DAPI was used for nuclei staining (blue). Data are shown as merged images. Original magnification x200 ( A, B ) and x900 ( C-F ).
jws-hep.217751.doc49KSupporting Information file jws-hep.217751.doc

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